The E3 ligases are involved in regulating other proteins by covalent ligation to the 76 amino acid protein ubiquitin. This post-translational modification can result in altered conformation, altered activity, or degradation of the substrate protein. Thus, E3 ligases are effectors of a major means of post-translational modification of proteins in many species, including mammals. Ubiquitin-protein ligation requires the action of an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase. In humans, there are thought to be two E1 enzymes, 36 E2 enzymes and up to 1000 E3 enzymes. Two main domains have been found to function as E3 ligases: HECT and RING domains; the majority of predicted E3 ligases have a RING domain rather than a HECT domain. The RING domain proteins can be further subdivided into the canonical RING domain and the RING-related domains known as U box and PHD finger. While HECT domain ligases contain a 350-residue ligase domain that directly binds the activated ubiquitin molecule, RING finger ligases serve as a scaffold for spatially coordinating the ubiquitin source and the target molecule. Although the RING finger motifs show little homology to each other, the core conserved structure for these domains is a pattern of cysteine and histidine residues coordinating two zinc ions. The dipeptide boronic acid bortezomib (PS-341, Velcade) is a potent proteasome inhibitor, has selective anticancer activity in tumor cells and in mice and was recently approved for clinical use in multiple myeloma. Selective E3 ligase inhibitors would be preferable because they should be more selective and less toxic. Unlike other large protein families, such as proteases and kinases, there are few small molecule tools for modulating the functions of E3 ligases. Specific inhibitors and activators are valuable for defining functions and substrates of proteins. RNA interference and cDNA expression can also assist in illuminating functions of proteins, but precise spatial and temporal regulation of proteins is optimally performed with small molecule modulators. In addition, E3 ligase domains are frequently found within the context of a large protein with other functions. Small molecules, in contrast to RNAi knockdown or cDNA expression, can readily reveal the function of a single domain in a large multi-domain protein, expressed at endogenous, physiological levels. In addition, small molecules can sometimes be developed into therapeutic agents. In this application, we propose to screen for small molecules that inhibit MDM2 E3 ligase activity. [unreadable] [unreadable] [unreadable]